Throughout this application various publications are referred by arabic numerals to within brackets. The disclosures for these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The discovery of RNA molecules that possess enzymatic, self-cleaving activity (ribozymes) has provided a new way to artificially control gene expression (Foster & Symons, (1987) Cell, 49: 585-591). Ribozymes have been designed that contain nearly all of the sequences required for cleavage. For the hammerhead type the target RNA needs to contain only the sequence XUX with cleavage occurring 3' from XUX (Haseloff & Gerlach, (1988) Nature, (London) 334: 585-591; Perriman et al., Gene (1992) 113: 157-163). The high specificity and limited target requirement give these catalytic RNA molecules the potential for inhibiting viral pathogens and for regulating specific gene expression by interfering with transcription in a highly specific manner (Uhlenbeck, (1987) Nature (London) 328: 596-600; Haseloff & Gerlach, (1988) Nature, (London) 334: 585-591).
Several reports indicate that the hammerhead type of ribozyme functions in living cells. Cotten & Birnstiel (1989, EMBO J., 8: 3861-3866) and Cameron & Jennings (1989, Proc. Natl. Acad. Sci., USA 86: 9139-9143) reported ribozyme-mediated destruction and lowering of specific gene expression in Xenopus laevis oocytes and monkey (COS1) cells, respectively. Sarver et al. (1990, Science, 247: 1222-1225) showed that a ribozyme directed against HIV-1 gag RNA reduced p24 antigen expression in CD4.sup.+ HeLa cells. Recently, this line of study was extended to bacterial cells by showing that a ribozyme designed to cleave the integrase gene of HIV-1 is effective when transcribed from a plasmid in Escherichia coli. Integrase RNA was eliminated and integrase protein synthesis was blocked (Sioud & Drlica, (1991) Proc. Natl. Acad. Sci., USA 88: 7303-7307). Since ribozymes are effective in vivo, problems of ribozyme stability and delivery may now be addressed.
To interfer with tumour necrosis factor .alpha. (TNF-.alpha.) gene expression we have used cationic liposome-mediated transfection (Malone et al., (1989) Proc. Natl. Acad. Sci., USA 86: 6077-6081) to deliver a ribozyme directed against TNF-.alpha. into human promyelocytic leukaemia cells (HL60) and peripheral blood mononuclear cells (PBMNC). TNF-.alpha. plays an important role in many inflammatory rheumatic diseases (Shinmei et al., (1989) Sem. Arth. Rheum. 18 (suppl. 1) 27-32), and it modulates the expression of several proteins, including the class I antigens of the major histocompatibility complex (MHC) and cytokines such as interleukin 1 and interleukin 6 (Beutler & Cerami, (1988) Annu. Rev. Biochem. 57: 505-518 and (1989) Annu. Rev. Immunol. 7: 625-655). TNF-.alpha. also appears to be necessary for normal immune responses, but large quantities of it can produce destructive effects such as those seen in rheumatoid arthritis (Brennan et al., (1989) Lancet ii 244-247). In addition, TNF-.alpha. is the cytokine responsible for the induction of HIV-1 expression in ACH-2 cells (Rosenberg & Fauci, (1990) Immunol. Today 11: 176-180). TNF-.alpha. induces the production of cellular factors that bind to the NF-.kappa.B enhancer elements within the viral long terminal repeat sequences and thereby activates HIV-1 expression.
The effectiveness of catalytic RNA molecules is dependent on the stability of the mRNA in vivo. In comparison with the knowledge of DNA structural elements, little is known about mRNA stability elements. m-RNA half-lives range from less than 30 minutes for fibroblast interferon and c-fos to greater than 17 hours for .beta. globin. Most eukaryotic mRNAs are protected in cells from exonuclease attack by the 5' cap structure and the 3'poly(A) tail and poly(A) binding proteins. In addition, eukaryotic mRNAs have both 5' and 3' non-coding regions on either side of the coding region. The 5' non-coding region is involved in the rate of initiation of translation of the mRNA to protein. The 3' non-coding region serves to initiate the formation of the poly(A) and can act to stabilize mRNA. (Baralle, F. E., Int. Rev. of Cytology (1983) 81: 71-106.) In particular, 3' non-coding iron-responsive elements have been identified that modulate mRNA stability in the presence of iron. Another characterized motif is the AUUUA element responsible for the rapid degradation of some cellular mRNAs, particularly cytokine mRNAs. (Saini, K. S. et al., Mol. Cel. Biochem. (1990) 96: 15-23; Ross, H. J. et al., Blood (1991) 77: 1787-1795). Some have postulated that an initial endonuclease attack is required, before rapid degradation can take place (Nielson, D. A. and Shapiro, D. J., Mol. Endocrinology (1990) 4: 953-957).
There is a need for methods to extend the half-life of particular mRNAs in vivo for protein production and oligonucleotide methods of gene control (antisense and triple helix) for use in plants and animals. Further, stabilizing mRNA elements can be applied to ribozymes in addition to antisense oligonucleotides.